As proteinas humanas FEZ1 e FEZ2 (fasciculation and elongation protein zeta) sao ortologas da proteina UNC-76 de C. elegans e estao envolvidas no crescimento e na fasciculacao dos axonios atraves de interacoes que envolvem kinesinas, mitocondrias e vesiculas sinapticas. Alem disso, algumas evidencias sugerem a participacao de FEZ1 na etiologia da esquizofrenia, no ciclo viral, alem da resistencia a quimioterapicos. Sua estrutura intrinsecamente desordenada, com coiled-coil ao longo da sequencia, pode contribuir para sua funcao. Nos exploramos a evolucao molecular da familia de proteinas FEZ com enfase no ramo dos vertebrados. Atraves do perfil do interactoma comparado entre FEZ1 e FEZ2 de Homo sapiens e UNC-76 de C. elegans foi observado um padrao de conservacao das interacoes proteinaproteina entre FEZ1 e UNC-76, que explicam a capacidade de FEZ1 resgatar os defeitos causados por mutacoes em unc-76 em nematoides, de acordo com o descrito por Bloom e colaboradores em 1997. Alem disso, caracterizamos a interacao entre FEZ1 e SCOCO (short coil-coiled) por SAXS (Small Angle X-ray Scattering). Essa interacao ja foi descrita previamente entre os seus ortologos UNC-76 e UNC-69, que cooperam no crescimento axonal. Um estado de heterotetramerico foi observado, consistindo de duas moleculas GST-SCOCO interagindo com duas moleculas de 6xHis-FEZ1 dimerizadas. Por PAGE (Polyacrylamide Gel Electrophoresis, eletroforese em gel de poli-acrilamida), SAXS, Espectrometria de Massas e Ressonância Magnetica Nuclear, constatamos que FEZ1 dimeriza envolvendo a formacao de ponte dissulfeto. In vivo, este estado dimerico de forma covalente pode ser importante para o transporte mediado por kinesinas de proteinas ao longo dos microtubulos. Assim, FEZ1 pode ser classificada como uma proteina adaptadora do transporte, dimerica e bivalente, essencial para o crescimento axonal e organizacao pre-sinaptica normal e transporte de cargas. A agregacao de novos parceiros de interacao encontrada para a proteina FEZ2 poderia ser interpretada como aquisicao de novas funcoes moleculares e pode ter ocorrido nos primeiros estagios da evolucao dos cordados
Abstract
Ki-1/57 is a 57-kDa cytoplasmic and nuclear protein associated with protein kinase activity and is hyper-phosphorylated on Ser/Thr residues upon cellular activation. In previous studies we identified the receptor of activated kinase-1 (RACK1), a signaling adaptor protein that binds activated PKC, as a protein that interacts with Ki-1/57. Here we demonstrate that the far-UV circular dichroism spectrum of the WD repeat-containing RACK1 protein shows an unusual positive ellipticity at 229 nm, which in other proteins of the WD family has been attributed to surface tryptophans that are quenchable by N-bromosuccinimide (NBS). As well as NBS, in vitro binding of 6×His-Ki-1/57(122–413) and 6×His-Ki-1/57(264–413) can also quench the positive ellipticity of the RACK1 spectrum. We generated a model of RACK1 by homology modeling using a G protein β subunit as template. Our model suggests the family-typical seven-bladed β-propeller, with an aromatic cluster around the central tunnel that contains four Trp residues (17, 83, 150, 170), which are likely involved in the interaction with Ki-1/57.
Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2-4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.
Additional file 1: Table S1. Proteins identified in keratoconus and control group tears. Gene names, protein names and ID, and Student´s t-test results comparing all proteins identified in keratoconus and control group.
Background: We recently performed metabolite profiling in 2,346 non-diabetic participants in the Framingham Offspring cohort. The glutamine/glutamate ratio was among the strongest predictors of the development of type II diabetes mellitus (DMII) during a 12-year follow-up period. Glutamate and glutamine are in a precursor-product relationship via the enzyme glutamine synthase (GS). Hypothesis: We hypothesized that inhibition of systemic GS activity would decrease the circulating glutamine/glutamate ratio and worsen glucose homeostasis in vivo. Methods: We administered 20 mg/kg of methionine sulfoximine (MSO), an irreversible inhibitor of GS, to 12 - 16 week old C57Bl6 mice intraperitoneally (i.p.) over 14 days (every other day for 7 injections). We then performed an intraperitoneal glucose tolerance test (IPGTT) with glucose measurements at 0, 30, 60, 90, 120 min from the tail vein. In a separate cohort of mice, we administered a single dose of 300 mg/kg of glutamine to both MSO-treated and control mice to assess reversibility of MSO effects. Results: Administration of MSO decreased circulating glutamine levels (n = 18, -64 ± 19% from baseline, p < 0.01), whereas glutamate levels increased significantly (n = 18, +25 ± 8, p = 0.03). The glutamine/glutamate ratio decreased from 2.16 ± 0.16 to 0.44 ± 0.03 (n = 12, p < 0.001). In the MSO-treated mice, the area under the curve of glucose excursion during the IPGTT increased markedly (n = 24, 12,098 ± 452 vs. 8,158 ± 368 AU, p < 0.001). In livers isolated from MSO-treated animals, we documented reduced liver GS activity by 94 ± 3% (n = 24, p < 0.001). Further, glutamine treatment of the MSO-treated animals at a dose of 300 mg/kg i.p. reversed the diabetogenic effects of MSO treatment (n = 12, 12,687 ± 568 vs 9,235 ± 399 AU, p < 0.01). Conclusion: Inhibition of systemic GS causes a reversal of the plasma glutamine/glutamate ratio and worsening of glucose tolerance. Glutamine administration rescues MSO-induced glucose intolerance. These studies implicate glutamine/glutamate metabolism in the control of glucose homeostasis. Further studies will focus on the mechanism of MSO-induced glucose intolerance and relate the murine findings to human pathology.
Identification of changes on the salivary protein profile (SPP) induced by the conditioning regimen (CR) for allogeneic hematopoietic stem cell transplantation (HSCT) can represent a helpful tool for diagnosis and management of the oral acute effects related with the HSCT. It was a prospective cohort involving 16 adult patients who underwent a first allo-HSCT for malignant hematological diseases. The condition regimen as well as the GVHD prophylaxis was performed according with Institutional Protocol for each underlying diseases. Unstimulated whole salivary samples were collected in two different periods: immediately before initiation of the conditioning regimen (A) and at day D+8 post-HSCT (B). Hyposalivation was clinically evaluated in both periods. Oral mucositis (OM) severity was graded according to WHO-criteria from D+8 until a complete engraftment. Salivary protein identification was performed through mass spectrometry (MS) analysis. A total of 65 proteins were identified in the collection A and 57 in the collection B. 39/65 (60%) salivary proteins were common founded in both collection. 7/39 (18%) showed differences (P < .05) among the two collections. The proteins Prolactin-Inducible Protein and Sthaterin were correlated with hyposalivation at D+8. The proteins Immunoglublin J Chain, Pulative uncharacterized protein DKFZp686N02209 and Statherin were correlated with Grade III-IV oral mucositis. These results suggested the rule of the conditioning regimens on damaging major salivary glands, leading to salivary proteomics changes. Hence, the proteomic changes were correlated with the severity of oral mucositis and hyposalivation. Those salivary proteins could represent a potential candidate of salivary proteomic biomarker panel to correlate with acute oral changes related with the HSCT. Granted by FAPESP (Sao Paulo Research Foundation), Brazil.
Lista de Abreviações e Siglas6xHis -cauda de 6 histidinas em proteínas recombinantes APPL -Amyloid precursor protein-like cDNAcomplementary DNA (DNA complementar) CLASP2 -CLIP-associating protein
Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs.We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1β, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation.Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor.This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases.
Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.
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